Effect of actinidin from kiwifruit juice and flavourzyme on the production of blue mussels extracts with high antioxidant activity

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1 Proceedings of the 2014 International Conference on Food Properties (ICFP2014) Kuala Lumpur, Malaysia, January 24 26, 2014 Effect of actinidin from kiwifruit juice and flavourzyme on the production of blue mussels extracts with high antioxidant activity Alaa El-Din Bekhit a, Jinzi Quinna Xu b, Alan Carne a a University of Otago, New Zealand b United States Abstract The effects of adding proteases (crude actinidin from Kiwifruit juice and commercial flavourzyme), incubation temperature (25, 35, 45 and 55 C) and incubation time (2, 4 and 6 hr) on the ph, total soluble content, colour parameters, protein hydrolysis profile and DPPH scavenging activity of blue mussels (Mytilus edulis aoteanus) were investigated in the present study. Higher total soluble content and better colour properties resulted from the use of kiwifruit juice compared with Flavourzyme. Flavourzyme and kiwifruit juice were effective in hydrolyzing the blue mussel protein at all incubation temperatures (Figure 2) with Flavourzyme being more effective than actinidin at 25 C. Blue mussel extracts produced by the proteases had very high and equivalent DPPH radical scavenging activity. The use of actinidin/ kiwifruit juice may be advantageous due to low cost and better sensory attributes. Keywords Hydrolysis, enzymes, antioxidant, shellfish 1. Introduction New Zealand blue mussels (Mytilus edulis aoteanus) are generated as a by-product of the well-known New Zealand Greenshell mussel farming. Blue mussel has similar composition and nutritional profiles as the Greenshell mussel, and it has been consumed either as a fresh or a processed product in many European and Asian countries for a long period of time [1-3]. Tonnes of blue mussels are harvested in New Zealand every year but have never been considered for commercial use [4]. Fermentation is a traditional way to preserve blue mussels in Asian countries [2, 5] and high antioxidant activities have been reported for the resultant liquid/sauce. However the conventional fermentation process takes up to 12 months to complete. For a long time proteases have been used in the food industry to accelerate food transformation. Proteases extracted from natural plants and other commercial enzymes have shown remarkable outcomes in various applications such as meat tenderization and fish protein extraction [6 8]. The purpose of this study was to use New Zealand blue mussel meat to produce mussel extracts under various processing conditions using a commercial enzyme [Flavourzyme], and actinidin (obtained from crude kiwifruit juice) and investigate the capability of the products to scavenge DPPH radical activity, as an indicator of antioxidant activity. 2. Materials and Methods 2.1 Sample Preparation New Zealand blue mussels (45 kg) were obtained from a commercial mussel farm in Invercargill (South Island), and shipped under chilled conditions to the University of Otago. The mussels were frozen at -30 o C soon after arrival and kept at this temperature until use (within 3 weeks of arrival). Mussels were thawed at 4 o C overnight before processing. The intact blue mussels were weighed, then opened with a shape knife and the mussel meat was removed and placed into a clean pre-weighed container to calculate the yield. Mussel meat was rinsed with distilled water, and excess moisture was allowed to run through a kitchen metal sieve. The mussel meat was minced using a Food Processor (Breville, Australia) at Speed 1 for 30 seconds. Minced mussel meat was weighed into 36 x 150 g aliquots in 500 ml beakers and 12 aliquots were allocated to kiwifruit juice treatment, 12 to Flavourzyme treatment and 12 to non-treated control. For kiwifruit juice treatment, fresh clear kiwifruit juice prepared as described by Han et al. (2009) was added (% w/w) to the samples. For Flavourzyme treatment, a commercial enzyme Flavourzyme (Novozyme, Denmark) was added (0.02% w/w) to the mussel meat. Each treatment group of 12 samples was divided into 4 groups of 3 which were assigned to different incubation temperatures (25, 35, 45 and 55 C) for time

2 course analysis of 2 hr, 4 hr and 6 hr incubation. Following the incubation step, samples were mixed with distilled water and made up to 500 ml, and then were heated in a 0 C boiling water bath for 1 hour, while covered with glass watches to minimize evaporation. After heating, the extracted material was separated from the solids and the volume reduced to 0 ml by evaporation. The mussel extracts were then cooled to room temperature, covered with plastic food wrap and stored at 4 C. 2.2 Analytical methods ph Measurement The ph values of all processed samples as well as raw mussel meat were measured in triplicate using a HANNA PH211 Microprocessor PH Meter (HANNA Instruments, Woonsocket, RI, USA). Total Soluble Content Total soluble content (Brix ) was measured by 0-32% Hand Refractometer (ATAGO Co., Ltd, Tokyo, Japan). Colour Measurement The colour of mussel sauce samples was measured using a HunterLab MiniScan XE Plus (Hunter Associates Laboratory, Inc., Reston, VA, USA). The MiniScan colorimeter is normally used to measure the colour of solid materials. After a consultation with a Hunter Associates representative, it was ascertained that the MiniScan could be used in this application. The MiniScan was initially calibrated with white and black standard tiles with an empty clean covered glass Petri dish placed on the top of the colour standards. For sample measurement, a covered Petri dish that was filled with the mussel extract sample was placed on top of the white standard tile and the colour reading was carried out. Measurements were performed in triplicate and the accuracy of measurements was validated using synthetic colour standards over several time points. Lightness (L*), redness (a*) and yellowness (b*) values were recorded and used for the calculation of hue angle (h) and chroma (C). Total Protein Determination The total protein content of the diluted mussel extracts was determined by Lowry s method. The protein concentrations in the samples were calculated from a standard curve constructed using bovine serum albumin. Samples were analyzed in triplicate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) The SDS-PAGE was performed on NuPAGE Novex Bis-Tris 4-12% mini gels (Life Technologies, Invitrogen, Carlsbad, California). Samples (150 ul) were subjected to a clean-up procedure to remove any lipids using a 2-D Clean-Up Kit (GE Healthcare, New Zealand) and re-dissolved in 90 ul NuPAGE LDS 1x sample buffer and μl of NuPAGE x reducing agent. The mixture was heated for min in a heating block at 90 C. The samples (6 μl) were loaded into the gel lanes and the gel was run at 160 V for 70 min. The gel was washed three times (5 min for each wash) in Milli-Q water with gentle agitation and then stained with Invitrogen Simply Blue stain followed by destaining with Milli-Q water. Free radical scavenging activity on α, α-diphenyl-β-picrylhydrazyl (DPPH ) assay The DPPH scavenging activity was determined as described by Brand-Williams et al. [9]. Each extract was diluted 300 fold and the resultant solution was tested for DPPH scavenging activity at serial dilutions of 0, 50, 25, 12.5, 6.25, and % (v/v), corresponding to 1, 2, 4, 8, 16, 32 and 64 fold dilution respectively. A sample extract solution (75 µl) was added to 2925 µl of g/l DPPH solution, prepared in 0% methanol and the mixture was mixed by inversion. The decrease in absorbance at 515 nm was determined initially (time zero), then after min and then after 1 hr incubation at room temperature using a UV/Visible spectrophotometer (Ultraspec 3300pro, Biochem Ltd., Cambridge, England) against water. The lower the absorbance of the reaction mixture corresponds to a higher DPPH radical scavenging activity. All determinations were performed in triplicate. The activity was compared with three of the most commonly used standard antioxidants (trolox, gallic acid and Butylated hydroxytoluene [BHT]) starting at a concentration of mg in the assay (i.e. dilution 1). 2.3 Statistical Analysis All results were obtained in triplicate and reported as mean ± standard deviation. Significant differences between means and multiple comparisons between means were analyzed by the GLM protocol. Analysis of variance

3 (ANOVA) was used to examine the effects of enzyme, incubation temperature, incubation time and their interactions on the measured parameters. Significant differences among the means were determined using the Tukey multiple comparison test in the GLM protocol at a confidence level of 95% (P < 0.05). 3. Results and Discussion Total soluble content (Brix ) The total soluble content was increased (P < 0.05) in the enzyme-treated samples compared to control (Figure 1). The highest total soluble content was obtained from Kiwifruit-treated blue mussels at 45 and 55 C. Flavourzymetreated mussels had the highest Brix produced at 35 C. samples had similar Brix at 35, 45 and 55 C which were higher than those obtained at 25 C. The Brix of the blue mussel extracts was increased by increasing the incubation time (P < 0.05). The highest Brix was obtained from kiwifruit juice-treated samples at 45 C after 6 hr. Most of the soluble components in fish sauces are free amino acids and small peptides []. Actinidin is particularly effective in hydrolyzing collagen [7] and was found to be effective in hydrolyzing collagen and proteins of salmon fillet [11]. Total soluble content (brix Kiwifruit Juice Flavourzyme Total soluble content (brix Kiwifruit Juice Flavourzyme Incubation temperature ( C) Incubation time (hr) Figure 1: Effect of proteases (actinidin from kiwifruit juice and flavourzyme), incubation temperature (25, 35, 45 and 55 C) and incubation time (2, 4 and 6 hr) on the total solids [Brix ] of blue mussel extracts ph The effects of treatments (control, kiwifruit juice and Flavourzyme), incubation temperature (25, 35, 45 and 55 C) and incubation time (2, 4 and 6 hours) on ph values of the produced mussel extract are shown in Table 1. Flavourzyme mussel extracts had the highest ph values, followed by kiwifruit juice treatment. The ph of the extracts was significantly higher (P < 0.05) in samples incubated for 2 hr compared to samples incubated for 4 and 6 hr and increased with increasing the incubation temperature. Overall, the mean ph values were influenced significantly by treatments and incubation time. Park et al. [2] found that amino acids can be released from the muscle of salted blue mussels by hydrolysis during fermentation, and the amino acid contents increased as the fermentation period increased. This was the result of proteolysis caused by microbial activity during fermentation and by the endogenous proteases. The addition of Flavourzyme to squid muscle can gradually increase the proteolysis of the squid muscle [12], and the increase in α-amino content was reported to indicate the extent of proteolysis [13]. The high ph values in Flavourzyme treated samples were likely caused by the release of basic amino acids. Higher ph was found in kiwifruit juice treated samples at temperature 45 C where actinidin has maximum activity. Colour parameters The impact of enzyme treatments, incubation times and incubation temperatures on the colour parameters of the mussel extracts is shown in Table 1. Generally, kiwifruit juice-treated samples had lighter colour and lower redness and yellowness components of the colour compared with control and Flavourzyme-treated samples (P < 0.05) whereas control samples had the highest values of these parameters. This led to the lowest colour intensity (i.e. chroma value) and the highest hue angle (h) indicating that the colour of the kiwifruit-treated samples was light redbrown. The lightness values of the control samples were increased while the redness values were decreased as the incubation temperature increased. The h-values of kiwifruit juice-treated samples were increased with the increase 0

4 of incubation temperature but the opposite correlation was found with Flavourzyme-treated samples, suggesting the importance of protease choice for obtaining a desired colour of mussel extract. There was no effect with incubation temperature on h-values of control samples. The reported darkening of fish sauce might be explained by the reaction of amine with aldehyde or ketone group [14], via the well-known Maillard reaction, which is believed to play an important role in color and flavor development of fish sauce during the ripening step. However, this justification is not plausible in the present study since kiwifruit juice, the treatment with the highest reduced sugars, resulted in the lightest and least red colour production. Table 1: Effect of proteases (actinidin from kiwifruit juice and Flavourzyme), incubation temperature (25, 35, 45 and 55 C) and incubation time (2, 4 and 6 hr) on the ph and colour parameters on blue mussel extracts. ph L* a* b* C h Treatment 5.61c 28.33c 16.15a 37.87a 41.25a 66.58c Kiwifruit 5.79b 37.12a 5.96c 25.14c 25.99c 77.13a Flavourzyme 6.03a 32.35b 9.48b 32.71b 34.09b 73.85b Treatment x Temp 25 C 5.59h 26.71i 16.04b 39.06a 42.28b 67.28e 35 C 5.58h 27.93h 17.31a 40.59a 44.23a 66.64e 45 C 5.63g 29.20g 15.63c 36.37b 39.68c 66.28e 55 C 5.63g 29.20g 15.62c 35.46b 38.80c 66.12e Kiwifruit 25 C 5.76f 36.27bc 5.88i 23.44f 24.28h 76.06b Kiwifruit 35 C 5.77f 36.97b 6.34h 24.30ef 25.20gh 76.48b Kiwifruit 45 C 5.80e 36.09c 6.78g 26.92d 28.04f 75.95b Kiwifruit 55 C 5.83d 39.13a 4.82j 25.91de 26.45fg 80.00a Flavourzyme 25 C 6.07a 34.73d 7.88f 31.72c 32.70e 76.30b Flavourzyme 35 C 6.05a 31.35f.16d 35.13b 36.62d 73.88c Flavourzyme 45 C 6.03b 32.51e 9.62e 31.53c 32.98e 72.82cd Flavourzyme 55 C 5.97c 30.79f.24d 32.47c 34.06e 72.38d Treatment x Time - 2 h 5.75d 31.09e 17.14a 43.87a 47.11a 68.52f - 4 h 5.54e 31.00e 17.21a 31.93e 36.80c 61.68g - 6 h 5.53e 22.9f 14.b 37.80b 40.35b 69.54f Kiwifruit - 2 h 5.81c 42.74a 1.89f 22.2h 22.29g 85.04a Kiwifruit - 4 h 5.74d 35.16b 7.85e 27.99f 29.09e 74.34bc Kiwifruit 6 h 5.81c 33.44c 8.12e 25.24g 26.59f 72.01e Flavourzyme - 2 h 6.11a 33.45c 8.04e 29.32f 30.44e 74.81b Flavourzyme - 4 h 5.99b 31.67de 9.86d 33.59d 35.03d 73.49cd Flavourzyme 6h 6.00b 31.92d.53c 35.23c 36.80c 73.24d Protein degradation profile Flavourzyme and actinidin, in kiwifruit juice, were effective in hydrolyzing blue mussel proteins at all incubation temperatures (Figure 2) with Flavourzyme being more effective than actinidin at 25 C (Figure 2A). Complete degradation of higher molecular proteins (bands > 55 kda) was achieved by these proteases and the efficacy of hydrolyzing these proteins was increased as the incubation time and the incubation temperature were increased. Maximum protein hydrolysis was achieved at temperatures 45 C for both enzymes as indicated by the low abundance of higher molecular weight proteins. Generally speaking, as previously reported [2], the addition of exogenous enzymes caused an increase in the protein content and decreased the carbohydrate content in fish sauce throughout the fermentation process. The levels of amino acids such as glycine, alanine, proline, aspartic acid and glutamic acid were higher in enzyme-added products, which may be important for the taste of fish and shellfish sauce [2]. It has been reported that the α-amino content increased during fermentation of samples due to the proteolytic activity of endogenous digestive enzymes and added commercial proteinases [13]. In conventional fermentation processes, it has been reported that an increase of soluble peptides extensively takes place during the

initial stages of fermentation, especially within the first 13 weeks of fermentation and becomes minimal thereafter [13].

A kda 160 1 80 60 50 40 30 20 15 B 3.5 kda C 160 1 80 60 50 40 30 20 15 3.

The treatments were carried at 25 C (A), 35 C (B), 45 C (C) and 55 C (D). lanes; 1)MW marker, 2) C 2 hr, 3) F 2 hr, 4) K 2 hr, 5) C 4 hr, 6) F 4 hr, 7) K 4 hr, 8) C 6 hr, 9) F 6 hr, ) K 6 hr.

The DPPH scavenging activity of extracts obtained at 45 C only are shown in Figure 3. Absorbance at 515 nm 0.7 0.6 0.5 0.4 0.3 0.2 0.

5 initial stages of fermentation, especially within the first 13 weeks of fermentation and becomes minimal thereafter [13]. The use of proteases from Koji soya bean and Flavouzyme can accelerate this proteolysis process. The present results are in agreement with the expected actions of proteases. A kda B 3.5 kda C D Figure 2: SDS-PAGE of protein extracts from blue mussel treated with kiwifruit juice (K), Flavourzyme (F), or nontreated control (C). The treatments were carried at 25 C (A), 35 C (B), 45 C (C) and 55 C (D). lanes; 1)MW marker, 2) C 2 hr, 3) F 2 hr, 4) K 2 hr, 5) C 4 hr, 6) F 4 hr, 7) K 4 hr, 8) C 6 hr, 9) F 6 hr, ) K 6 hr. DPPH radical scavenging activity The blue mussel extracts demonstrated very high DPPH scavenging activity even after 300-fold dilution (Figure 3). The DPPH scavenging activity of extracts obtained at 45 C only are shown in Figure 3. Absorbance at 515 nm A B C Dilution factor Kiwifruit juice treated Flavourzyme treated Trolox Gallic acid BHT Absorbance at 515 nm Dilution factor Kiwifruit juice treated Flavourzyme treated Trolox Gallic acid BHT Absorbance at 515 nm Dilution factor Kiwifruit juice treated Flavourzyme treated Trolox Gallic acid BHT Figure 3: DPPH scavenging activity of extracts (45 C and 6 hr incubation only) from blue mussel treated with kiwifruit juice, Flavourzyme, or non-treated control. The figures are for 0 time (A), min reaction time (B) and 1hr reaction time (C).The activity of standard antioxidants (gallic acid, trolox and BHT) at starting at a concentration of mg in the assay. blue mussel extracts had weak antioxidant activity compared with Flavourzyme and kiwifruit juice hydrolyzed samples. The activity was slightly lower than gallic acid at 0 and minutes of reaction time but was

6 identical after 1 hr of reaction time. The DPPH scavenging activity of hydrolyzed blue mussels was better than trolox and BHT. High antioxidant activity of fermented blue mussel sauce has been reported [5, 15]. The radical scavenging activity of round Scad protein hydrolysates prepared using Alcalase or Flavourzyme produced extracts with higher DPPH radical scavenging activity [16]. Actinidin from kiwifruit is a comparable protease to flavourzyme and can produce extracts with superior sensory attributes. 4. Conclusion Blue mussel extracts produced with Flavourzyme and kiwifruit demonstrated very high radical scavenging activity. Both protease preparations had similar activity which was comparable to gallic acid and better than trolox and BHT. The results indicate that blue mussel extracts have potential as a flavouring ingredient containing antioxidant activity and have potential use in health promoting applications. References 1. Kyriazi-Papadopoulou, A., Vareltzis, K., Bloukas, J.G., and Georgakis, S., 2003, Effect of Smoking on Quality Characteristics and Shelf-life of Mediterranean Mussel (Mytilus galloprovincialis) Meat Under Vacuum in Chilled Storage, Italian Journal of Food Science, 15(3), Park, P.J., Je, J.Y., and Kim, S.K., 2005, Amino Acid Changes in the Korean Traditional Fermentation Process for Blue Mussel, Mytilus edulis, Journal of Food Biochemistry, 29(1), Turan, H., Sonmez, G., Celik, M.Y., Yalcin, M., and Kaya, Y., 2007, Effects of Different Salting Process on the Storage Quality of Mediterranean Mussel (Mytilus Galloprovincialis L. 1819), Journal of Muscle Foods, 18(4), McLean, C.H., and Bulling, K.R., 2005, Differences in Lipid Profile of New Zealand Marine Species Over Four Seasons, Journal of Food Lipids, 12(4), Rajapakse, N., Mendis, E., Jung, W.K., Je, J.Y., and Kim, S.K., 2005, Purification of a Radical Scavenging Peptide from Fermented Mussel Sauce and its Antioxidant Properties, Food Research International, 38(2), Ha, M., Bekhit, A.E.D., Carne, A., and Hopkins, D.L., 2013, Comparison of the Proteolytic Activities of New Commercially Available Bacterial and Fungal Proteases Toward Meat Proteins, Journal of Food Science, 78(2), C170-C Ha, M., Bekhit, A.E.D., Carne, A., and Hopkins, D.L., 2013, Characterization of Kiwifruit and Asparagus Enzyme Extracts, and Their Activities Toward Meat Proteins, Food Chemistry, 136(2), Han, J., Morton, J.D., Bekhit, A.E.D., and Sedcole, R., 2009, Pre-rigor Infusion with Kiwifruit Juice Improves Lamb Tenderness, Meat Science, 82(3), Brand-Williams, W., Cuvelier, M.E., and Berset, C., 1995, Use of a Free Radical Method to Evaluate Antioxidant Activity, Food Science and Technology, 28(1), Hjalmarsson, G.H., Park, J.W., and Kristbergsson, K., 2006, Seasonal Effects on the Physicochemical Characteristics of Fish Sauce Made from Capelin (Mallotus villosus), Food Chemistry, 3(2), Aspmo, S.I., Horn, S.J., and Eijsink, V.G.H., 2005, Enzymatic Hydrolysis of Atlantic Cod (Gadus morhua L.) Viscera, Process Biochemistry, 40(5), Xu, W., Yu, G., Xue, C.H., Xue, Y., and Ren, Y., 2008, Biochemical Changes Associated with Fast Fermentation of Squid Processing By-Products for Low Salt Fish Sauce, Food Chemistry, 7(4), Yongsawatdigul, J., Rodtong, S., and Raksakulthai, N., 2007, Acceleration of Thai Fish Sauce Fermentation Using Proteinases and Bacterial Starter Cultures, Journal of Food Science, 72(9), M382- M Lopetcharat, K., and Park, J.W., 2002, Characteristics of Fish Sauce Made from Pacific Whiting and Surimi By-Products During Fermentation Stage, Journal of Food Science, 67(2), Jung, W.K., Rajapakse, N., and Kim, S.K., 2005, Antioxidative Activity of a Low Molecular Weight Peptide Derived from the Sauce of Fermented Blue Mussel, Mytilus edulis, European Food Research and Technology, 220 (5-6), Thaiansilakul, Y., Benjakul, S., and Shahidi, F., 2007, Antioxidative Activity of Protein Hydrolysate from Round Scad Muscle using Alcalase and Flavourzyme, Journal of Food Biochemistry, 31 (2),

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